For acting enzymes on deoxyribonucleic acid (DNA) transcription and replication, the double stranded DNA must be cleaved by helicase firstly. Topoisomerase could cut double-stranded supercoiled DNA to change the DNA conformation, then connecting the original cleavage sites of the DNA. The topoisomerase comprises topoisomerase I (Top I) and topoisomerase II (Top II). The Top I cuts one strand of double-stranded supercoiled DNA via destroying phosphodiester bonds to form a small nick. In the meanwhile, the other strand of the double-stranded supercoiled DNA can pass through the nick for re-ligation. Both strands of double-stranded DNA can be cleaved by the Top II and then religated by changing the DNA conformation.
At the rapid multiplication phase of cells, the high concentration and hyperactivity of the topoisomerase are presented. Thus, anti-cancer drugs can be developed by pharmacists via inhibiting the topoisomerase activity to inhibit or block the growth of cancer cells at slow or rapid multiplication phase. The topoisomerase activity must be relied on during the life cycles of immune cells or pathogenic infection sources, such as virus, bacteria or protozoa. If there are inhibitors to interfere the topoisomerase activity, the life of the aforesaid cells can be affected. Previous studies have indicated that topoisomerase inhibitors are applied to anti-tumor, anti-infection sources, whose infection sources are such as virus, bacteria and etc., anti-inflammatory and immune regulation fields. There is effort to study in the synthesis and research of the topoisomerase inhibitors in the pharmaceutical industry.
Traditionally, the topoisomerase inhibitors is analyzed by a gel electrophoresis or cell responses, but the drawbacks thereof are time consuming. Alternatively, the enzyme inhibitors are analyzed by a 3D computer-aided molecular modeling. The results obtained from the 3D computer-aided molecular modeling do not represent real enzyme responses, and thus the obtained results are inconsistent with the results of the actual enzyme activity. Recently, the topoisomerase inhibitor activity can be effectively detected by chip techniques. Using the chip techniques for screening topoisomerase inhibitors can increase greatly performance. Especially, the micro-fluid chip development can be more convenient and rapid for studying in the dynamic analysis of the topoisomerase inhibitors. Before the present invention is disclosed, substances immobilized onto chips are DNA or inhibitors, and topoisomerase is used as a mobile phase and injected into the chip immobilized with DNA or inhibitors to be analyzed. However, the results thereof are often to cause large consumption of the topoisomerase. Additionally, steps and important notices for purifying the topoisomerase having catalytic activity are more complicated than those of DNA. When the topoisomerase is used as the mobile phase, the experiment results must be considered with variables during purifying the topoisomerase. The catalytic activity of proteins is not easy to maintain, and the proteins are degraded easily, such that the screening purpose is not reached quickly. Furthermore, a DNA molecule often has a plurality of biding sites for the topoisomerase, and the topoisomerase has different affinities on different biding sites so as to increase the variables with regard to determine affinity constants, which results in analyzing incorrectly while testing the dynamic analysis. A person having ordinary skill in the art hopes to effectively resolve the difficulty.